No abstract

Background: ␣-Synuclein aggregates cause early neurite pathology by as yet unknown mechanisms. Results: ␣-Synuclein oligomers and seeds decrease microtubule stability, kinesin-microtubule interaction, cellular cargo distribution, and neurite network morphology. Conclusion: Various ␣-synuclein species interact differently with proteins of axonal transport. Significance: The impairment of the microtubule-kinesin function by ␣-synuclein oligomers drives early neurite pathology.

Conventional kinesin is a motor protein which translocates organelles from cell centre to cell periphery along specialized filamentous tracks, called microtubules. The direction of translocation is determined by microtubule polarity. This process of biological force generation can be simulated outside cells with kinesin-coated particles actively moving along immobilized microtubules. The in vitro approaches of kinesin-mediated transport described so far had the disadvantage that concerning their polarity the microtubules were randomly distributed resulting in random transport direction. The present paper demonstrates the unidirectional translocation of kinesin-coated cargoes across arrays of microtubules aligned not only in a geometrically parallel but also in an isopolar fashion. As cargo, glass, gold, and polystyrene beads with diameters between 1 and 10 µm were used. Independent of material and size, these beads were observed to move unidirectionally with average velocities of 0.3-1.0 µm s-1 over distances up to 2.2 mm. Moreover, the isopolar microtubule arrays even enabled the transport of large flat glass particles with an area of up to 24 µm×12 µm and 2-5 µm thickness which obviously contacted more than one microtubule. The controlling transport direction is considered to be an essential step for future developments of motor protein-based microdevices working in nanometre steps.

Kinesin is a microtubule-associated protein, converting chemical into mechanical energy. Based on its ability to also work outside cells, it has recently been shown that this biological machinery might be usable for nanotechnological developments. Possible applications of the kinesin-based motor system require the solution of numerous methodological and technical problems, including the orientation of force generation into a desired direction and the determination of the tolerable roughness of the surfaces used, the minimal free vertical space still enabling force-generating activity, and the temporal stability of the system. This paper reports on the example of microtubules gliding across kinesin-coated surfaces and shows that the force-generating system needs a minimal free working space of about 100 nm height and works up to 3 h with nearly constant velocity. Individual microtubules were observed to cover distances of at least 1 mm without being detached from the surface and to overcome steps of up to 286 nm height. In addition, mechanically induced flow fields were shown to force gliding microtubules to move in one and the same direction. This result is regarded as being an essential step towards future developments of kinesin-based microdevices as this approach avoids neutralization of single forces acting in opposite directions.

DeCuevas et al. [J. Cell Biol. 116 (1992) 957^965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30³C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27³C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45³C; at higher temperatures, kinesin was irreversibly damaged.z 2000 Federation of European Biochemical Societies.